The traditional view of insulin release machinery in pancreatic ? cells relies essentially on the influx of Ca2+ from the extracellular medium, eventually leading to insulin secretion. The mechanisms involved in Ca2+ mobilization from the major intracellular storage compartment, which in metazoan cells is represented by the endoplasmic reticulum (ER), remain less understood. The main intracellular Ca2+ release channels are Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs). The role of RyR in the pathophysiology of type 2 diabetes mellitus (T2DM) has been recently clarified, demonstrating that RyR is crucial in glucose-stimulated insulin secretion. Instead, the exact role of ? cell IP3R in T2DM is poorly understood and remains a glaring knowledge gap in the metabolic field. Three isoforms of IP3R have been identified in mammals (IP3R1-3); their expression pattern is overlapping and functionally redundant. Pancreatic ? cells express all IP3R isoforms, and their levels are upregulated by chronic glucose stimulation. We hypothesize that IP3Rs play a key role in the pathophysiology of T2DM. In the present proposal, we will test this hypothesis in vivo, ex vivo, and in vitro using state-of-the-art models including both genetic and pharmacologic tools. Scientific premise and rationale: Genome-based studies in humans have demonstrated that gain-of-function mutations in genes encoding for IP3Rs are linked to perturbations in glucose homeostasis and enhanced susceptibility to diet-induced diabetes; similarly, genetic mapping identified IP3Rs as a risk factor for T2DM; however, these associations have not been functionally explained. Moreover, controversial findings have been reported when attempting to examine the actual role of IP3Rs in ? cells. We have robust preliminary data showing that IP3Rs are significantly upregulated in islets from T2DM patients compared with non-diabetic individuals; similarly, we detected a marked upregulation of IP3Rs in islets from mice fed high-fat diet (HFD) and db/db mice compared with non-diabetic littermates fed standard chow. On these grounds, we will explore the following specific aims: Aim 1 will define in vivo the functional role of ? cell IP3Rs in the pathogenesis of T2DM, characterizing the metabolic phenotype of a novel, ? cell-specific, animal model, whereas Aim 2 will identify the molecular mechanisms linking IP3Rs to pancreatic ? cell (dys)function in T2DM, focusing on mitochondrial fitness and autophagy. Importantly, although the ER-mitochondrial interface is known to be a primary site for autophagosome formation, the exact role of IP3Rs in autophagy and mitophagy remain extremely debated. We will conduct assays in mice, in islets, and in ? cells; human islet studies are included to investigate potential similarities and differences between murine and human ? cells. We also designed rescue studies to verify if the proposed mechanisms are necessary and sufficient to mediate the effects of IP3Rs in ? cells. The planned experiments are highly relevant, with a high degree of conceptual and technical innovation; indeed, our studies will provide an unbiased assessment of the functional role of ? cell IP3Rs and define previously unrecognized pathways linking IP3Rs, mitochondrial dysfunction, and autophagy/mitophagy in the pathophysiology of T2DM.